Flavin Amine Oxidases from the Monoamine Oxidase Structural Family Utilize a Hydride Transfer Mechanism



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The amine oxidase family of enzymes has been the center of numerous mechanistic studies because of the medical relevance of the reactions they catalyze. This study describes transient and steady-state kinetic analyses of two flavin amine oxidases, mouse polyamine oxidase (PAO) and human lysine specific demethylase (LSD1), to determine the mechanisms of amine oxidation. PAO is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the oxidation of N1-acetylated polyamines. The pH-dependence of the kcat/Kamine indicates that the monoprotonated form of the substrate is required for catalysis, with the N4 nitrogen next to the site of CH bond cleavage being unprotonated. Stopped-flow spectroscopy shows that the pH-dependence of the rate constant for flavin reduction, kred, displays a pKa of 7.3 with a decrease in activity at acidic pH. This is consistent with an uncharged nitrogen being required for catalysis. Mutating Lys315 to methionine has no effect on the kcat/Kamine-pH profile with the substrate spermine, and the kred value only shows a 1.5-fold decrease with respect to wild-type PAO. The mutation results in a 30- fold decrease in kcat/KO2. Solvent isotope effects and proton inventories are consistent with Lys315 accepting a proton from a water molecule hydrogen-bonded to the flavin N5 during flavin oxidation. Steady-state and transient kinetic studies of para-substituted N,N'-dibenzyl-1,4- diaminobutanes as substrates for PAO show that the kred values for each correlate with the van der Waals volume (VW) and the value. The coefficient for VW is the same at pH 8.6 and 6.6, whereas the p value increases from -0.59 at pH 8.6 to -0.09 at pH 6.6. These results are most consistent with a hydride transfer mechanism. The kinetics of oxidation of a peptide substrate by human lysine specific demethylase (LSD1) were also studied. The kcat/KM pH-profile is bell-shaped, indicating the need for one unprotonated nitrogen next to the site of CH bond cleavage and another protonated nitrogen. The kcat and kred values are equal, and identical isotope effects are observed on kred, kcat, and kcat/KM, indicating that CH bond cleavage is rate-limiting with this substrate.